In 2013, the World Health Organization (WHO) revised the current HIV treatment and prevention guidelines and emphasized the importance of HIV viral load (VL) testing in the management of HIV positive patients. Instead of clinical presentations and CD4 cell counts, WHO strongly recommends using HIV viral load testing to monitor the HIV Antiretroviral Therapy (ART) and dropped the cutoff of the level of HIV plasma VL from 5000 copies/mL to 1000 copies/mL for therapeutic efficacy. In other words, if HIV patients under ART have a viral titer in plasma is greater than 1000 copies/mL, it will be considered as a drug treatment failure. Responses to treatment failure, such as adherence counseling, drug resistance testing and second line treatment regimens, are time-consuming and expensive, and should be used only if necessary.
After infection, HIV virus not only starts to replicate itself in the infected cells, but also integrates its cDNA into the host chromosomes as the latent HIV proviral DNA (Greene, W. C. and Peterlin, B. M. 2002. Charting HIV's remarkable voyage through the cell. Nat Med. 8(7): 673-80; Blankson, J. N., D. Persaud, R. F. Siliciano. 2002. The Challenge of Viral Reservoirs in HIV-1 Infection. Annu. Rev. Med. 53:557-593). HIV viral load is normally measured using plasma as a specimen type. However, in resource limited settings, such as Africa, plasma samples may not be easily obtained, stored, transferred, and tested (World Health Organization. 2013. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. World Health Organization, Geneva). Dried blood spots (DBS) have been evaluated as a solution for HIV viral load testing in the resource limited settings, with limited success (Smit P W et al. 2014. Systematic review of the use of dried blood spots for monitoring HIV viral load and for early infant diagnosis. PLoS ONE. 9(3): e86461; Bertagnolio, S., N. T. Parkin, M. Jordan, J. Brooks, J. G. Garcia-Lemia. 2010; Dried blood spots for HIV-1 Drug Resistance and Viral Load Testing: A Review of Current Knowledge and WHO Efforts for Global HIV Drug Resistance Surveillance. AIDS Rev. 12:195-208; Johannessen, A. 2010; Dried blood spots in HIV monitoring: applications in resource-limited settings. Bioanalysis. 2(11):1893-1908). Roche Molecular Diagnostics (RMD) has a research use only (RUO) product, developed in 2009, which uses the real-time PCR COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) HIV-1 Test v2.0 and Dried Fluids Spot Procedure (DFSP), to measure the HIV viral load in DBS. Unfortunately, when paired plasma and DBS samples are tested, the DFSP test yields higher viral load titers compared to the plasma gold standard. This overestimation is particularly pronounced in samples with plasma viral loads less than 5,000 copies/mL, presumably due to the detection of cell-associated HIV DNA and RNA.
According to the CAP/CTM HIV-1 DFSP procedure, HIV DBS is first incubated with a chaotropic agent—Specimen Pre-Extraction (SPEX) buffer and the total nucleic acids, including HIV viral RNA in blood fluids, plus other cell-associated HIV DNA and RNA, such as HIV proviral DNA, are extracted and eluted from DBS into the SPEX buffer. Then, the extracted buffer is placed on the CAP/CTM instrument for nucleic acid purification followed by HIV target amplification and detection. The currently existing nucleic acid extraction methodologies for HIV DBS VL measurement fully lyse all cells and denature all protein complexes, allowing the complete extraction of total nucleic acids from DBS, including free HIV viral particles in blood fluids and cell-associated HIV RNA and DNA.
Over-quantification of HIV VL in DBS is a problem not only with Roche's DBS assay, but also observed with other commercially available assays, such as Abbott's HIV DBS assay. In HIV scientific and medical communities, it has been speculated that HIV DNA present in HIV infected cells in the DBS causes the over quantification of the viral load, although the exact mechanisms of this over quantification phenomenon are not clear or demonstrated (Médecins Sans Frontieres Access Campaign, 2013). The overestimation in DBS may be ameliorated but not eliminated by amplification procedures that have specificity for RNA over DNA (e.g., the NASBA procedure used by BioMerieux NucliSENS®). There remains a need for alternative procedures for addressing the challenges of HIV VL over-quantification in a sample.